Alzheimer's disease (AD) is the most prevalent form of dementia. It is a neurodegenerative disorder, clinically characterized by progressive loss of memory and general cognitive function, and pathologically characterized by the deposition of extracellular proteinaceous plaques in the cortical and associative brain regions of sufferers. These plaques mainly comprise fibrillar aggregates of beta-amyloid peptide (Aβ). Aβ is formed from amyloid precursor protein (APP). APP is a ubiquitous membrane-spanning (type 1) glycoprotein, of which three major isoforms (APP695, APP751, and APP770; SEQ ID NOS:1-3, respectively) are known, that undergoes a variety of proteolytic processing events (Selkoe, 1998, Trends Cell Biol. 8:447-453). APP in general is disposed with its N-terminal portion in the extracellular space or in the lumen of an intracellular organelle such as the Golgi or an endosome, a transmembrane portion, and its C-terminal portion extends into the cytosol (Thinakaran and Koo, 2008, J. Biol. Chem. 283:29615-19619).
Generation of Aβ from APP occurs via separate intracellular proteolytic events involving the enzymes beta-secretase and gamma-secretase. Beta-secretase (also called BACE1) first cleaves APP within the extracellular domain to shed the soluble N-terminal APP-sbeta or sAPPβ (SEQ ID NO:5), leaving the beta-CTF (C-terminal fragment; SEQ ID NO:8) membrane-bound. The latter is then further processed by gamma-secretase to release Aβ and gamma-CTF, or AICD (APP intracellular domain). Given that gamma-secretase cleaves beta-CTF, beta-CTF has widely been used to monitor gamma-secretase activity in cell based and in vitro assays. The gamma-secretase cleavage site of APP is situated within a transmembrane domain; indeed gamma secretase represents one of small number of proteases that act within a membrane milieu. (See FIG. 1 for a schematic diagram of proteolytic cleavages of APP leading to Aβ and other polypeptide products.) Variability in the site of gamma-secretase mediated proteolysis results in Aβ peptides of varying chain lengths comprising heterogeneous C-termini, e.g. Aβ (1-38, “Aβ38”; SEQ ID NO:15), Aβ (1-40, “Aβ40”; SEQ ID NO:16) and Aβ (1-42, “Aβ42”; SEQ ID NO:17). (See FIG. 9A for the portion of the APP sequence containing the cleavage sites of the various secretase activities.) After secretion into the extracellular medium, the initially-soluble Aβ forms soluble oligomeric aggregates, ultimately resulting in the insoluble deposits and dense neuritic plaques which are one of the pathological hallmarks of AD. Aβ42 is more prone to aggregation than Aβ40 and is the major component of amyloid plaque (Jarrett, et al., 1993, Biochemistry 32:4693-4697; Kuo, et al., 1996, J. Biol. Chem. 271:4077-4081).
Alternatively, APP can be sequentially cleaved by alpha-secretase and gamma-secretase to produce soluble APP-alpha, or sAPPα (SEQ ID NO:4), P3 and gamma-CTF (FIG. 1). Alpha-secretase cleavage occurs at a site distinct from the cleavage site of beta-secretase and precludes the formation of Aβ peptides.
Various interventions in the plaque-forming process have been proposed as therapeutic treatments for AD (see, e.g., Hardy and Selkoe, 2002, Science 297:353-356). One such method of treatment that has been proposed is that of blocking or attenuating the production of Aβ, for example, by inhibition of beta- or gamma-secretase. Other proposed methods of treatment include administering a compound(s) which blocks the aggregation of Aβ, or administering an antibody which selectively binds to Aβ. Activation of α-secretase is also an appealing strategy for the development of AD therapy, in that increased alpha-secretase cleavage might lend to lessened Aβ generation.
Gamma-secretase is a macromolecular proteolytic complex composed of at least four proteins: presenilin (PS), nicastrin (NCT), PEN-2 and APH-1 (De Strooper, 2003, Neuron 38:9-12). Recently, CD147 and TMP21 have been found to be associated with the gamma-secretase complex (Chen, et al., 2006, Nature 440:1208-1212; Zhou et al., 2005, Proc. Natl. Acad. Sci. USA, 102:7499-7504). Among these known components, PS is believed to contain the active site of gamma-secretase, recognized as an aspartyl protease (Esler et al., 2000, Nat. Cell. Biol., 2:428:434; Li et al., 2000, Nature 405:689-694; Wolfe et al., 1999, Nature 398:513-517). Considerable effort has been made to understand the process of gamma-secretase substrate recognition and its catalytic machinery. A PS-dependent protease can process any single-pass transmembrane (TM) protein regardless of its primary sequence as long as the TM protein extracellular domain is smaller than 300 amino acids. Moreover, the size of the extracellular domain appears to determine the efficiency of substrate cleavage (Struhl and Adachi, 2000, Mol. Cell. 6:625-636).
The sequential cleavage of APP by two proteases (beta- or alpha-secretase followed gamma-secretase) is analogous to a recently defined signaling paradigm, known as regulated intramembrane proteolysis (RIP) (Brown et al., 2000, Cell 100:391-398). RIP generally requires two proteolytic steps to initiate its signaling cascade, whereby the second intramembrane cleavage is dependent on the first cleavage. Indeed, Notch, a type I transmembrane protein, employs RIP and is a substrate for gamma-secretase cleavage. It has been determined that γ-secretase cleaves a multitude of other substrates that include the Notch receptors (Kopan R, Goate A: A common enzyme connects Notch signaling and Alzheimer's disease. Genes Dev 2000, 14(22):2799-2806), ErbB-4 (Kopan R, Goate A: A common enzyme connects notch signaling and Alzheimer's disease. Genes Dev 2000, 14(22):2799-2806), CD44 (Lammich S, Okochi M, Takeda M, Kaether C, Capell A, Zimmer A K, Edbauer D, Walter J, Steiner H, Haass C: Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide. J Biol Chem 2002, 277(47):44754-44759), as well as the Notch ligands Delta-1 and Jagged-2 (Six E, Ndiaye D, Laabi Y, Brou C, Gupta-Rossi N, Israel A, Logeat F: The Notch ligand Deltal is sequentially cleaved by an ADAM protease and gamma-secretase. Proc Natl Acad Sci USA 2003, 100(13):7638-7643; Ikeuchi T, Sisodia S S: The Notch ligands, Deltal and Jagged2, are substrates for presenilin-dependent “gamma-secretase” cleavage. J Biol Chem 2003, 278(10):7751-7754; and others). Deregulated Notch signaling has been associated with the development of various cancers, including T-cell Acute Lymphoblastic Leukemia (T-ALL) (Weng A P, Ferrando A A, Lee W, Morris J Pt, Silverman L B, Sanchez-Irizarry C, Blacklow S C, Look A T, Aster J C: Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 2004, 306(5694):269-271). As such, inhibitors of gamma-secretase activity might not only have implications in the treatment of AD, but may also have benefit in treatment of all diseases in which gamma-secretase plays a role.